Figure 5
From: Structure and mutagenesis of the DNA modification-dependent restriction endonuclease AspBHI
S41A and S41C activity assays on methylated oligonucleotide substrates.
(a) Schematic diagram of the fully methylated oligonucleotide substrates (M = 5mC) used for analyzing possible cleavage products (P1–P5 shown in panel b). (b) Duplex oligonucleotides (20 ng) were incubated at 37°C for 2 hours with 0.5 μg (0.29 pmoles) of WT, S41A, or S41C. Products were resolved on a 20% TBE native PAGE gel and visualized with Sybr Gold staining. Inserted is a 10–20% gradient SDS-PAGE showing the proteins used for crystallization (Se-Met) and for activity (WT, S41A and S41C). NEB protein ladder was used as molecular weight markers.
